Bacteriorhodopsin (BR) is a 27,000 dalton protein that can be isolated from the purple membrane of Halobacterium halobium. It functions as a light driven proton pump. Crystalline purple membrane fragments are isolated, solubilized by detergent and reconstituted into pure egg yolk lecithin vesicles. Tryptophan fluorescence reveals a highly blue shifted emission spectrum whose quantum yield is unchanged with light-dark adaptation. Tryptophan lifetime heterogeneity analysis reveals two populations of fluorophores, one of which is altered by light adaptation. Fluorescence energy transfer from BR tryptophan to anthrostearic acid probes inserted into the vesicles allows us to map the spatial distribution of the tryptophans and sensitively monitor conformational changes in the protein. This is a new technique that will allow in situ conformational analysis of membrane proteins. Studies of the detailed pumping kinetics of BR reconstituted into lipid vesicles has allowed us to model the processes involved in pumping of protons.